Project Summary ? Project 3 The development of alcohol use disorder (AUD) results from an interaction between both environmental and genetic factors. Studies of the impact of environment on the risk to develop AUD have, to date, focused mainly on psychological characteristics (externalizing phenotypes, etc.) or on developmental defects associated with prenatal or adolescent exposure to alcohol. Here, we will study the role of the dietary omega-3 fatty acid eicosapentaenoic acid (EPA), an environmental factor, in adults for a role in modulating the acute behavioral response to ethanol. Our study of the alcohol-naive acute level of response (LR) is significant because this measure is strongly correlated with the liability to develop AUD. Using two model organisms (C. elegans and mouse), we have previously found that dietary omega-3 fatty acid levels significantly impact the acute LR to alcohol in ethanol-naive animals. In worms, we have found specifically that EPA is essential for the development of acute functional tolerance (AFT) to ethanol. We will use two complementary approaches to identify the mechanisms underlying the effects of EPA on the acute behavioral response to ethanol. In Aim 1 we will identify genes whose expression is regulated in response to different EPA levels (depleted, normal levels, above normal levels) in adult C. elegans. We will correlate gene expression changes and the time course of the effect of dietary EPA on AFT. We will directly test the roles of prioritized candidate genes in the development of AFT to identify the molecular pathways that are important for ethanol response behaviors and are affected by EPA levels. In Aim 2, we will use cutting edge lipidomics to determine what lipid species are derived from the dietary EPA. We will correlate the accumulation of EPA derived lipids to the time course of the effects of dietary EPA on AFT. We will identify candidate lipid mediators of the development of AFT. This will implicate molecular pathways in the machinery underlying AFT. These studies will provide novel insight into the roles of lipids in regulating the level of response to alcohol. Determination of the nature of these roles will enable future identification of the protein targets of ethanol, and the regulatory mechanisms affecting those proteins, that are impacted by these lipid-dependent functions. Finally, in Aim 3, we will continue a successful approach that uses the high-throughput genetic power of C. elegans to test the hypothesized roles of genes that have been implicated by other components of the VCU-ARC in behavioral responses to ethanol.